The immunoglobulin G (IgG) class of antibodies plays an important role in adaptive immune defense of the human host against pathogens. IgG consists of two identical heavy chains and two identical light chains, which in turn are composed of variable and constant domains. Papain treatment of IgG molecule can generate Fab fragments recognizing antigens and Fc fragment, a recognition site for host receptors and a site of interaction with a number of effector molecules.
The Fc portion of IgG also contains a conserved complex carbohydrate or glycan attached to the asparagine 297 residue in the CH2 domain of each heavy chain. These glycans are located in the interface between the CH2 domains when IgG is in its native form. They consist of a biantennary core of N-acetylglucosamine and mannose with added terminal and branching carbohydrate residues such as N-acetylglucosamine, fucose, sialic acid, and galactose. The presence of this carbohydrate is crucial for proper antibody structure and interactions with cellular immunoglobulin G Fcγ receptors (FcγRs) and the complement system.
Endoglycosidase S (EndoS) is secreted by Streptococcus pyogenes and has a specific endoglycosidase activity on native IgG by hydrolyzing the conserved glycans attached to the asparagine 297 residue on the heavy chains of IgG. EndoS is the first known bacterial enzyme with a unique specificity for native IgG. In contrast, the activities of other known endoglycosidases require or are enhanced by denaturation of the glycoprotein substrate.
Antibodies such as IgG have many applications in basic research as well as in diagnostics and drug development. In some of these applications, such as immunohistochemistry, immunoassays, tumour detection, radiotherapy, crystallographic studies of antibody binding sites and immunotargeting, it is more convenient to use Fab fragments than whole IgG molecules. Some of the advantages of using Fab fragments are that they will not be affected by Fc receptors on cells or precipitate antigen, they display a reduced immunogenicity and are less susceptible to phagocytosis, and that radiolabelled Fab fragments are more rapidly cleared from tissue than whole IgG molecules. For other applications, it is desirable to use Fc fragments of IgG.
If Fab or Fc fragments are to be produced on a large scale they may be produced as recombinant proteins. For purification purposes, recombinant IgG and serum-produced IgG is often produced as a whole molecule and then chemically processed to obtain Fab or Fc fragments.
The cleavage of IgG into Fab and Fc fragments is most often carried out using proteolytic enzymes such as pepsin or papain. These enzymes often cleave other proteins, so the cleavage reaction generally has to be performed on a purified IgG fraction. Furthermore, pepsin and papain typically cleave IgG in more than one place. This means that the fragments obtained often do not correspond to whole Fab or Fc fragments, and even if cleavage does result in Fab and Fc fragments, they are typically susceptible to further cleavage into smaller fragments. The isolation of Fc fragments from Fab fragments is most often carried out using protein A or G affinity separation columns, which utilise the Fc-binding properties of the bacterial proteins A and G.